High Density Growth of T7 Expression Strains with Auto-Induction Option

ABSTRACT

A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

PARENT CASE TEXT

This application is a continuation of U.S. application Ser. No.11/741,282, filed Apr. 27, 2007, which is a continuation-in-part of U.S.application Ser. No. 10/675,936 filed Sep. 30, 2003, which applicationdraws priority of U.S. Provisional Application No. 60/455,032, filedMar. 14, 2003.

GOVERNMENT SUPPORT

This invention was made with Government support under contract numberDE-AC02-98CH10886, awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA sequencing projects have provided coding sequences for hundreds ofthousands of proteins from organisms across the evolutionary spectrum.Recombinant DNA technology makes it possible to clone these codingsequences into expression vectors that can direct the production of thecorresponding proteins in suitable host cells. The resulting proteinsare widely useful, as objects of biochemical, biophysical, structuraland functional studies for understanding basic biological processes, asenzymes to serve as research tools or produce valuable chemicals, asdiagnostics, vaccines, therapeutics or targets for developing medicallyuseful drugs, or for protein chips, to mention a few.

The T7 expression system comprises in vivo inducible expression, in T7expression system host strains, of T7 RNA polymerase from a chromosomalcopy of a cloned gene for the T7 RNA polymerase enzyme (gene 1 ofbacteriophage T7), followed in turn by recognition and binding of a T7promoter sequence contained in T7 expression vectors carried in the hoststrain, followed by intense transcription of any gene(s) cloneddownstream of the T7 promoter sequence, and, where the cloned sequenceis a protein coding sequence, subsequent translation of the transcripts.This recombinant gene expression system, originally developed inEscherichia coli and which has become the standard by which otherprokaryotic expression systems are judged, has been adapted for use inother bacterial species including Salmonella enteric serovar Typhimurium(McKinney, J., et al. (2002) J. Bacteriology 184:6056-6059), Pseudomonas(Schweizer, H P (2001) Curr. Opin. Biotechnol. 12:439-445), Rhodobactercapsulatus (Drepper, T., et al. (2005) Biochem. Soc. Trans. 33:56-58),Ralstonia eutropha (Barnard, G. C., et al. (2004) Prot. Exp. & Purif.38:264-271) and Bacillus subtilis (Conrad, B., et al. (1996) Mol. Gen.Genet. 250:230-236).

The inducible T7 expression system is highly effective and widely usedto produce RNAs and proteins from cloned coding sequences in thebacterium Escherichia coli (Studier and Moffatt, J. Mol. Biol. 189:113-130 (1986); Studier et al., Methods in Enzymology 185: 60-89 (1990);Novagen). The coding sequence for T7 RNA polymerase is typically presentin the chromosome under control of the inducible lac or lacUV5 promoterin the chromosome of host cells such as BL21(DE3), B834(DE3) andHMS174(DE3), and derivatives of such cells such as ER2566 and ER 2833(New England Biolabs). In another derivative strain, designated“BL21-AI” (Invitrogen), gene 1 is under the control of thearabinose-inducible araBAD promoter. In the absence of inducingcompounds, transcription by the host cell RNA polymerase is blocked bythe natural endogenous repressor. In the case of the lac promoter, it isthe lac repressor and for the araBAD promoter, the product of the araCgene is the repressor.

The coding sequence for the desired RNA or protein (referred to as thetarget RNA or protein) is typically placed in a plasmid under control ofa T7 promoter, that is, a promoter recognized specifically by T7 RNApolymerase. In the absence of an inducer for the lacUV5 promoter, littleT7 RNA polymerase or target protein should be present and the cellsshould grow well. However, upon addition of an inducer, typically IPTG(isopropyl-β-D-thiogalactoside), T7 RNA polymerase will be made and willtranscribe almost any DNA controlled by the T7 promoter. T7 RNApolymerase is so specific, active and processive that the amount oftarget RNA produced can be comparable to the amount of ribosomal RNA ina cell. Thus, large amounts of RNAs that are useful in themselves, suchas ribozymes, can be produced. If the target RNA contains the codingsequence for a protein and appropriate translation initiation signals(such as the sequence upstream of the start codon for the T7 majorcapsid protein), target protein can be produced, often accumulating tobecome a substantial fraction of total cell protein. See also U.S. Pat.Nos. 4,952,496; 5,693,489; and 5,869,320, the contents of which areincorporated herein by reference.

In strains in which the T7 gene 1 (T7 RNA polymerase gene) is undercontrol of the lac or lacUV5, IPTG has typically been used to induceexpression of target proteins. Lactose will also cause induction and,being much cheaper than IPTG, may be preferable for large-scaleproduction. Neubauer et al., Appl. Microbiol. Biotechnol. 36: 739-744(1992) obtained induction by lactose with the same efficiency as withIPTG by careful monitoring of the glucose level in fermentation and byaddition of lactose when the glucose was nearly depleted. Hoffman etal., Protein Expression and Purification 6: 646-654 (1995) used similarprocedures to obtain comparable levels of protein synthesis with lactoseor IPTG induction in a fermentor process.

A problem in using inducible T7 expression systems is that T7 RNApolymerase is so active that a small basal level can lead to asubstantial expression of target protein even in the absence of addedinducer. If the target protein is sufficiently toxic to the host cell,establishment of the target plasmid in the expression host may bedifficult or impossible, or the expression strain may be unstable oraccumulate mutations (Kelley et al., Gene 156: 33-36 (1995)). Aneffective means to reduce basal expression (and thereby increase therange and stability of target proteins that can be established andexpressed) is to place the lac operator sequence (the binding site forlac repressor) just downstream of the start site of a T7 promoter,creating a T7lac promoter (Dubendorff and Studier, J. Mol. Biol. 219:45-59 (1991)). Lac repressor bound at the operator sequence interfereswith establishment of an elongation complex by T7 RNA polymerase at aT7lac promoter and substantially reduces the level of target mRNAproduced. If sufficient lac repressor is present to saturate all of itsbinding sites in the cell, the basal level of target protein inuninduced cells is substantially reduced, but induction unblocks boththe lacUV5 and T7lac promoters and leads to the typical high levels ofexpression. Thus, the T7lac promoter increases the convenience andapplicability of the T7 system for expressing a wide range of proteins.

It was early noticed that growth of T7 expression cultures to saturationcould cause problems, and Grossman et al., Gene 209: 95-103 (1998)showed that cultures growing in certain complex media induce the targetprotein to high levels upon approach to saturation even when the T7lacpromoter was used. They pointed out that such unintended induction couldbe a problem in isolating and using strains that express proteins thatare toxic to E. coli. They concluded that the known inducer lactose wasnot responsible for this effect, but that cyclic AMP is required, andthey recommended using a mutant unable to make cyclic AMP as anexpression host.

Although such basal level of expression from T7 expression vectors canbe suppressed through use of the T7lac promoter in such vectors, thisdoes not solve problems resulting from unintended induction of T7expression strains when they are grown to saturation, which was noted byGrossman et al. (Gene 209: 95-103 (1998)). Grossman et al. showed thatcultures growing in certain complex media induce the target protein tohigh levels upon approach to saturation even when the T7lac promoter wasused. They pointed out that such unintended induction could be a problemin isolating and using strains that express proteins that are toxic toE. coli. In their work, they concluded that lactose had not beenresponsible for this effect, but that cyclic AMP was required, and theyrecommended using a mutant host strain that is unable to make cyclicAMP. They also found that addition of 1% glucose to late log phase cellsprevented the unintended induction, and the Novagen web site referencestheir paper and recommends adding 1% glucose to the medium to managethis problem.

Structural genomics is an area where multi-milligram amounts of manydifferent proteins over a wide evolutionary range are required fordetermination of protein structures by X-ray crystallography or nuclearmagnetic resonance (NMR). Fabrication of protein chips is anotherapplication where many different proteins are needed. Expressing clonedcoding sequences in the T7 system is an efficient, widely used methodfor obtaining these proteins. Screening large numbers of clones forprotein expression level and solubility makes it desirable to haveprocedures that can be applied to many clones in parallel, preferablyusing automation. The need to process many cultures in parallel dictatesbatchwise growth of cultures in small vessels such as culture tubes ormulti-well plates such as the 24-, 96- or 384-well plates commonlyavailable. A high level of protein production per volume of culture isalso desirable. The needed multi-milligram amounts of pure protein couldbe produced in fermentors, but cultures grown batchwise in vesselsaerated by shaking (a baffled flask on a rotary shaker, for example),bubbling air, or oxygen can typically produce this amount of protein inthe T7 expression system in a liter or less of culture, allowing severalcultures, each producing a different protein, to be grown and induced inparallel.

In trying to develop reliable procedures for growing and inducingprotein synthesis in many cultures in parallel, a significant difficultywas to obtain all of the cultures in a comparable state of growth sothat they could be induced simultaneously in parallel. Substantialeffort was required to measure the cell density of each culture and addinducer at the proper time, even using a plate reader that could measurethe densities of cultures in all of the different wells of a plate in asingle reading. Even if comparable amounts of culture could beinoculated in each well, differences in lag time or growth ratetypically generated situations where cultures in different wells wouldbe ready for induction at substantially different times. If the entireplate was to be collected at once, cultures would also vary in thelength of time in which they had been producing target protein, possiblymaking it difficult to choose a time when all had been induced tooptimal levels without substantial overgrowth of some cultures by cellsthat had lost plasmid.

An obvious strategy was to grow the entire plate to saturation in asmall volume of medium in each well, dilute by adding fresh medium, growfor an appropriate time (determined by previous testing or by directmeasurement of cell densities), and add inducer to all wells at the sametime. The hope was that all cultures in a plate would saturate at nearenough to the same density and grow after dilution with similar enoughkinetics that the culture-to-culture variation in density at the time ofinduction would be tolerable. However, in trying to implement thisstrategy, when certain lots of complex growth media were used, theproblem described by Grossman et al. (1998) was encountered, namely,induction during the growth to saturation. Indeed, it was found thatmedia made with a particular lot of N-Z-amine showed this inductionbehavior, whereas otherwise identical media made with a second lot fromthe same supplier did not. Unwanted induction at saturation would makeit extremely difficult to obtain sufficient uniformity of growth topermit parallel manipulation of cultures expressing target proteins ofdifferent, usually unknown degrees of toxicity. Although addition ofglucose could suppress this induction (Novagen), the saturated culturescould become very acid, which would limit the saturation density andagain make it difficult to get uniform growth upon dilution. Screeningdifferent lots of N-Z-amine for those without the inducing behavior didnot seem to be an attractive solution, as there was no guarantee thatsuch lots would always be available. Thus, the approaches taken, leadingto the present invention, were to determine causes of and ways toprevent unwanted induction and to develop means to promote desirableauto-induction of expression strains.

The ability to control the problem of sporadic, unwanted induction incomplex media would represent a significant advance in the art. Asystematic analysis of the components of both complex and defined mediawas undertaken. The goal was to define requirements for batchwise growthof T7 expression strains to high density under conditions suitable forgrowth and induction of many cultures in parallel, and, complementarily,to develop formulations that would reliably grow cultures of expressionstrains to saturation with little or no induction.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a method and growthmedia for promoting auto-induction of transcription of cloned DNA incultures of bacterial cells grown batchwise, the transcription beingunder the control of a promoter whose activity can be induced by anexogenous inducer whose ability to induce said promoter is dependent onthe metabolic state of said bacterial cells. Initially, a culture mediais provided which includes: i) an inducer capable of inducingtranscription from said promoter in said bacterial cells; and ii) ametabolite that prevents induction by said inducer, the concentration ofsaid metabolite being adjusted so as to substantially preclude inductionby said inducer in the early stages (early to mid-log phase) of growthof the bacterial culture, but such that said metabolite is depleted to alevel that allows induction by said inducer at a later stage of growth(mid to late-log phase and prior to saturation). The culture medium isinoculated with a bacterial inoculum, the inoculum comprising bacterialcells containing cloned DNA, the transcription of which is induced bysaid inducer. The culture is then incubated under conditions appropriatefor growth of the bacterial cells until such growth sufficientlydepletes the metabolite such that auto-induction of transcriptionoccurs, and where applicable the transcripts have been translated.

In a preferred embodiment, the present invention relates to promotingauto-induction of transcription of cloned gene 1 of bacteriophage T7 inbatchwise-grown bacterial cells, which cloned gene is under the controlof an inducible promoter and is stably propagated in the bacterialcells. A culture medium comprising an exogenous inducer which is capableof inducing transcription from the inducible promoter and one or moreconstituents that prevents induction by said inducer until such time asgrowth and division of the bacterial cells has depleted the one or moreconstituents of the culture medium to a level that permits induction bythe inducer. The provided culture medium is inoculated with thebacterial cells, the culture is incubated under conditions for growth ofthe bacterial cells until the level of the one or more constituents hasbeen depleted to a level permitting auto-induction of transcription ofthe cloned gene 1.

This embodiment of the present invention further includes incubating theculture to permit translation of the gene 1 transcripts into T7 RNApolymerase enzyme. The present invention also includes bacterial cellswhich are T7 expression strains, i.e., the cells contain a T7 expressionplasmid in which a target gene is under the control of a T7 promoter(e.g., pET vectors (Novagen); Variflex vectors (Stratagene) and pRSETvectors (Invitrogen), etc.). The present invention further includesincubating the culture until such time as the T7 RNA polymerase enzymehas transcribed target genes under the control of a T7 promoter, and,further includes incubating the culture until such time as the lattertranscripts are translated into protein. In general the preferredembodiment includes incubating the inoculated cultures until asaturating cell density is achieved.

In another aspect, the present invention relates to a method forimproving the production of a selenomethionine-containing protein orpolypeptide in a bacterial cell, the protein or polypeptide beingproduced by recombinant DNA techniques, the bacterial cell encoding avitamin B12-dependent homocysteine methylase. The method for improvingthe production of this protein or polypeptide includes culturing thebacterial cell in a culture medium containing vitamin B12.

In another aspect, the invention relates to a method for suppressingtranscription of cloned DNA in cultures of bacterial cells grownbatchwise, said transcription being under the control of a promoterwhose activity can be induced by an exogenous inducer whose ability toinduce said promoter is dependent on the metabolic state of saidbacterial cells. This aspect includes the steps of: a) providing aculture medium comprising a carbon source whose uptake and metabolism bysaid bacterial cells suppresses induction of transcription from saidpromoter; b) inoculating the culture medium with a bacterial inoculum,the inoculum comprising bacterial cells containing cloned DNA, thetranscription of which is suppressed by the carbon source; and c)incubating the culture of step b), with shaking, under conditionsappropriate for growth of the bacterial cells while suppressingtranscription of the cloned DNA.

DETAILED DESCRIPTION OF THE INVENTION 1. Development of Non-InducingMedia

Well known media for growth of E. coli and production of target proteinswith the T7 expression system include ZB, ZY (equivalent to LB), M9 orM9ZB, which contain various combinations of 1% of a tryptic digest ofcasein (such as tryptone or N-Z-amine AS), 0.5% yeast extract, 0.5%NaCl, or the components of M9 medium (Studier and Moffatt, J. Mol. Biol.189: 113-130 (1986)). These were the starting components in the searchfor formulations that would allow batchwise growth to high celldensities with reproducible and reliable behavior relative to inductionof protein expression in the T7 system. The E. coli strains used fortesting growth and expression were primarily BL21(DE3) or B834(DE3),alone or containing plasmids containing coding sequences under controlof the T7lac promoter and the upstream translation initiation signals ofthe T7 major capsid protein.

The standard measure of growth was optical density at 600 nm (A600)after dilution in water to concentrations that gave readings below 0.25.Viability and stability of cultures grown under different conditionswere tested by plating, usually on agar plates containing ZB. A standardconfiguration for testing different media formulations in parallel wasgrowth of 0.5 ml cultures in 13×100 mm glass culture tubes vertically ina plastic rack in a gyratory incubator at 300-350 rpm. The usualincubation temperature was 37° C., although 20° C. or even lowertemperatures were also tested. Time courses of more than a few pointswere measured in 125-ml Erlenmyer flasks, usually containing 5 ml orless of medium. These configurations provided sufficient aeration tosustain logarithmic growth to an A600 approaching 10 in the appropriatemedia. Higher levels of aeration could be achieved with smaller volumesof culture in larger vessels, but the above configurations were usedbecause results obtained with them seemed to translate well to 500 mlculture volumes in 1.8- or 2.8-liter baffled Fernbach flasks, convenientfor producing multi-milligram amounts of as many as six proteins at oncein a gyratory incubator.

Although they support good growth and protein expression, the usualmedia are far from optimal. In a typical experiment, saturation density(A600) in ZB was 1.2, in ZY was 2.8, in M9ZB without glucose was 2.6,and in M9ZB containing 2% glucose was 5.8. The usual glucose content ofM9ZB is 0.4%, and metabolism of the higher concentration overwhelmed thebuffering capacity of M9ZB, producing a final pH of 4.6. Increasing theconcentration of N-Z-amine in ZB increased the saturation densityapproximately in proportion to concentration up to at least 8%, whichsaturated at A600 of about 8. Adding 1% glucose to 8xZB or ZY gavelittle change in saturation density, but greatly reduced the final pH.Increasing the buffering capacity of the medium allowed saturation inglucose-containing media at densities greater than 10. Clearly, evencomplex media are limited for components needed for growth, andmaintenance of a pH near neutral is important for obtaining growth tohigh density.

To test induction of T7 RNA polymerase in expression hosts in theabsence of a target plasmid, the T7 deletion mutant 4107 was used(Studier and Moffatt, J. Mol. Biol. 189: 113-130 (1986)). This mutant T7phage lacks the entire coding sequence for T7 RNA polymerase and isunable to form a plaque on a lawn of cells unless the host cell suppliesT7 RNA polymerase. The basal level of T7 RNA polymerase in uninducedBL21(DE3) is low enough that only small plaques develop at lowefficiency, and they typically begin to appear only after about 3 hours.In contrast, induced BL21(DE3) supports large plaques that becomeapparent in less than 2 hours, typical of wild-type T7. This 4107 plaqueassay was used to test whether T7 polymerase was induced in cultures ofBL21(DE3) grown in different media. Cultures grown in media made withthe lot of N-Z-amine that did not give induction of target proteins atsaturation also appeared uninduced in the plaque assay, giving onlysmall plaques that took a long incubation time to develop. Culturesgrown in media made with the lot of N-Z-amine that did give induction oftarget proteins at saturation rapidly gave the large plaques indicativeof induction, unless the growth medium also contained glucose, whichappeared to prevent induction, as others had reported previously.

Fully defined media were formulated with simple salts and with glucoseas the sole carbon source, both to test the requirements for differentnutrients and to develop non-inducing media that would support growth ofT7 expression strains to saturation at reasonable densities with thelowest possible basal levels of T7 RNA polymerase. When all nutritionalrequirements are satisfied, the main limitation in achieving high celldensities appears to be maintaining the pH of the culture near neutral,since metabolism of glucose can produce substantial amounts of acid. Onesolution to this problem is to buffer the medium with phosphate, arequired nutrient that buffers in the neutral range. Increasingconcentrations of phosphate can buffer the acid generated by higherconcentrations of glucose, allowing higher cell densities to be attainedwhile maintaining a pH near neutral (typically greater than pH 6) allthe way to saturation. However, too much phosphate can be inhibitory,presumably because of high ionic strength or osmotic pressure. Aphosphate concentration of 100 mM seems a reasonable compromise,providing enough buffering capacity to allow growth to saturation in0.5% glucose while maintaining a pH close to neutrality. Such culturesgrow with a doubling time of about 60-70 minutes at 37° C. and saturateat A600 of approximately 5 to 6 and a pH around 6.5. One such growthmedium is P0.5G (Table 1).

Several other compounds besides phosphate were tested for ability tohelp manage the acid produced by the metabolism of glucose or glycerolduring growth to saturation. Succinate proved to be particularly useful.Growth of BL21(DE3) on a simple salts medium containing 75 mM succinate(0.23%) as sole carbon source was relatively slow (about a 2 hourdoubling time), but the ph of the culture increased with growth (to a pHof 8.7 at an A600 of 0.9). In glucose-succinate mixtures, culturesappear to metabolize glucose preferentially, doubling at about the samerate as in cultures where glucose is the sole carbon source. Atappropriate succinate and glucose concentrations, the pH of the growingculture initially decreases and then reverses, presumably as glucose isdepleted and succinate is metabolized. For a given medium and culturecondition, the range of succinate concentrations that balance the acidgeneration by glucose, glycerol, or other carbon sources whosemetabolism generates acid is easily determined empirically. With toolittle succinate, the pH at saturation may fall well below 5; with toomuch succinate the pH at saturation may increase beyond pH 9. In thepreferred range of succinate concentrations, the pH at saturation liesbetween 6.5 and 7.5. A culture grown in a simple salts medium containing0.5% glucose and 20 mM succinate as sole carbon sources (NIMS medium,Table 1), typically saturates at A600 of approximately 5 to 6, and pHbetween 6.5 and 7.5. Growth of BL21(DE3) in simple salts with succinateas sole carbon source did not appear to induce T7 RNA polymerase, asdetermined by the 4107 plaque assay.

Fumarate and DL-malate are cheap and readily available carbon sourcesthat behave like succinate in their ability to balance acid generation.Citrate and acetate are also effective, but somewhat less so. Growth ofBL21(DE3) in simple salts with succinate as sole carbon source did notappear to induce T7 RNA polymerase, as determined by the 4107 plaqueassay. Neither succinate nor any of these other carbon sources, inmixtures with glucose in simple salts media, appear to cause inductionof target protein synthesis in T7 expression strains.

Considerable testing of growth of BL21(DE3) or B834(DE3), with andwithout target plasmids, showed that all expression strains tested, evenstrains that express highly toxic target proteins such as the gene 7.7protein of bacteriophage T7, grow to saturation in P0.5G or NIMS mediumwith little or no expression of target protein, loss of plasmid, or lossof ability to express plasmid. These media can provide a substantialadvantage over typical lots of ZY (or LB) in obtaining expressionstrains for target proteins that are deleterious to the host. In thecase of T7 gene 7.7, transformants in BL21(DE3) readily gave colonies onP0.5G plates, but no colonies on ZY plates made with the lot ofN-Z-amine that caused induction upon approach to saturation.

The ability to balance acid generation with succinate or other carbonsources allows complete flexibility in testing the minimumconcentrations of all components (including phosphate) needed for growthof T7 expression strains (or any bacteria) and good expression of targetproteins. Tests with P0.5G, NIMS, and media with other combinations ofcarbon sources (Table 1) showed that minimum concentrations to assuregrowth to an A600 of at least 5 and good expression of target proteinsinclude approximately 0.5 mM Mg, 5 mM PO₄, 25 mM NH₄, 0.5 mM SO₄, 5-10μM Fe, and lower concentrations of other metal ions. At least 10 mM Mg,150 mM PO₄, 100 mM NH₄, 25 mM SO₄, and 500 μM Fe can be tolerated withlittle or no effect on growth to high density and expression of targetproteins. Concentrations in the media that have been tested mostextensively have been 1 or 2 mM Mg, 25-100 mM PO₄, 25-100 μM NH₄, 1-25mM SO₄, and 5-100 μM Fe (Table 1).

Since a significant fraction of proteins bind metals for stability orfunction, and in structural genomics or other projects the metal-bindingproperties of target proteins may be unknown, the mixture of trace metalions given in Table 2 was designed to provide most metal ions that areknown to be specifically bound by proteins. A target protein of 50,000Da produced at a level of 100 mg/liter would have a concentration of 2μM and a protein of 10,000 Da would have a concentration of 10 μM. The1× concentration of metal mix provides 2-50 μM of each of 10 differenttrace ions, probably sufficient to saturate most metal-binding proteinsthat would be produced. If the metal content of a target protein isknown, a saturating amount of that metal can be added specifically. Incultures where target protein is not to be expressed, 0.1× concentrationof metal mix should be sufficient for high-density growth, or 50 μM Feplus 0.02-0.05× metal mix. In cultures whose growth was limited by lackof trace metals, the most dramatic stimulations of growth upon theaddition of 1, 10 or 100 μM of individual metal ions of the metal mixwere provided by 10-100 μM Fe, 1-100 μM Mn and 1-10 μM Co. Evidence oftoxicity up to 100 μM concentration was seen only for Co, which caused alag before growing well at 10 μM and almost completely prevented growthat 100 μM, and Se, which supported growth normally at 10 μM, but onlypoorly at 100 μM. At least 5× concentration of metal mix had no apparentdeleterious effect on growth to high densities and expression of targetproteins in a simple salts medium, and at least 10× metals was toleratedin a simple salts medium plus 200 μg/ml each of 18 of the natural aminoacids (no cysteine or tyrosine).

BL21(DE3) has no nutritional requirements for growth. B834(DE3) wasknown to require methionine for growth, and 200 μg/ml is sufficient forhigh density growth and expression of target proteins. In the course ofthis work, it was discovered that the methionine requirement of B834could be satisfied instead by vitamin B12 (as little as 1 nM). Thisdemonstrated that B834 is a metE mutant, defective in the vitaminB12-independent homocysteine methylase that would normally synthesizemethionine, the last step in methionine biosynthesis. Vitamin B12 isknown to activate a second homocysteine methylase of E. coli, theproduct of metH. Since E. coli is unable to synthesize vitamin B12, thissecond enzyme is active only when this vitamin is added to the growthmedium. Ability to grow on either methionine or vitamin B12 is thedefining characteristic of metE mutants.

To make the media described here generally useful for the growth ofexpression hosts or other bacteria that may have additional, sometimesmultiple nutritional requirements, specific growth factors, mixtures ofgrowth factors such as amino acids, vitamins or nucleosides, or complexmedia components such as N-Z-amine and yeast extract may be added to theminimal media. Some such media are given in Table 1. A useful stocksolution is the mixture of 17 of the 20 natural amino acids, each at 10mg/ml. Left out of this mixture are cysteine, which slowly forms theessentially insoluble cystine and precipitates; tyrosine, which is onlyslightly soluble; and methionine, which is often used for radioactivelabeling or is replaced by selenomethionine (Se-Met) to label proteinsfor phasing X-ray crystallographic data. Addition of 18 amino acids (noC or Y) substantially increases the growth rate over that of simplesalts media, giving a doubling time of 30-35 minutes as opposed to 60-70minutes. A concentration of 200 μg/ml of each amino acid seemssufficient for most purposes, although higher concentrations may bebetter for very high density growths. The omission of cysteine andtyrosine seemed to have little effect on growth rate or saturationdensity compared to a mixture containing all 20 amino acids. In contrastto amino acids, addition of mixtures of vitamins or nucleosides seemedto give little if any stimulation of the growth rate or increase insaturation density of BL21(DE3), which has no specific requirement forthem. Addition of the complex ZY components to the minimal salts mediaseemed to provide slightly higher growth rates and saturation densitiesthan the addition of 18 amino acids in most experiments. To minimize thepossibility of induction near saturation in complex media, glucoseshould be added at a high enough concentration that it will not bedepleted (as indicated by a decrease in pH and no subsequent increase),typically 0.8% in ZYP medium.

Extensive experience with growing and storing cultures of BL21(DE3) andB834(DE3), alone or containing expression plasmids, in NIMS and in P0.5Gindicates that these are excellent media for stable storage of freezerstocks in 8% glycerol at −70° C. or working stocks stored in therefrigerator and used for inoculating subcultures. In contrast toprevious experiences with other media, cultures grown to saturation inthese media remain viable for periods of weeks to months of storage inthe refrigerator, retaining their titer (typically greater than 10¹⁰/ml)and ability to grow subcultures with little or no lag.

2. Development of Auto-Inducing Media

Hoffman et al., Protein Expression and Purification 6: 646-654 (1995)reported that fed-batch techniques in a well controlled fermentorallowed induction of target protein synthesis in the T7 system byaddition of lactose or glucose-lactose mixtures after depletion ofglucose. They proposed that these techniques may slow induction rate andallow more time for proper folding and solubility while producingamounts of protein comparable to those obtained by IPTG induction andculture densities with A600 in the range of 25-40.

It was of interest to determine whether such techniques, when applied tobatchwise production of proteins in the T7 expression system in thesenew media, could increase the solubility of target proteins that wereexpressed well, but were largely insoluble, of which several were in astructural genomics project. Also, studies of the ClpP protein of E.coli had revealed considerable variability in the fraction of solubleClpP produced, suggesting that lower rates of induction might produce alarger fraction of soluble protein.

The 4107 plaque assay confirmed that growth of BL21(DE3) to saturationin minimal medium containing 2% α-lactose (and no glucose), with orwithout added ZY, caused induction of T7 RNA polymerase. However, adding0.1% or 0.5% lactose to complex medium containing 2% glucose gave only aslight indication of a possible increase in T7 RNA polymerase in the4107 plaque assay.

Target protein P35 of a structural genomics project (yeast proteincoproporphyrinogen III oxidase) under control of a T7lac promoter in apET vector in B834(DE3) was expressed to substantial levels when grownto saturation in ZYP medium made with the lot of N-Z-amine that showedinducing activity. The protein is apparently not very toxic to theexpression host, because the plating efficiency of cells from thesaturated culture appeared to be normal. Addition of 1% lactose to themedium produced about a 50% increase in saturation density, but littleincrease in the level of target protein. However, almost all of thecells capable of forming a colony had lost plasmid. (It was soondiscovered that even cells without plasmid can grow quite well in ZYPmedium in the presence of 25 μg/ml of kanamycin, the concentration usedin this experiment.) Apparently, 1% lactose caused induction to such ahigh level that cells that contained plasmid were killed. Addition of0.05% or 0.1% glucose substantially increased the level of targetprotein produced, apparently by allowing growth to higher density beforethe level of induction caused by 1% lactose became high enough to killthe cells. Almost all of these surviving cells had also lost plasmid.

These results suggested that perhaps the induction in certain lots ofZYP medium was in fact due to the presence of a small amount of lactose,contrary to what Grossman et al., Gene 209: 95-103 (1998) concluded.Their conclusion was based on their finding that β-galactosidasetreatment of the medium did not eliminate the induction phenomenon andtheir inability to detect lactose in the medium (<0.002%). It was testedwhether adding lactose to ZYP medium made with the lot of N-Z-amine thatshowed no inducing activity could reproduce the observed behavior of thelot with inducing activity, namely induction of the target protein tolevels readily apparent by gel electrophoresis upon growth to saturationwith retention of a typical plating efficiency of cells that essentiallyall retained plasmid. Indeed, expression was detected by SDS gelelectrophoresis with Coomassie blue staining with as little as 0.001%added lactose in the non-inducing medium, and substantial induction wasobserved at 0.003% lactose or higher. Loss of titer in the saturatedcultures started to become significant at 0.01% to 0.02% lactose and wassevere by 0.05%. Thus, the lactose concentrations that can causedetectable induction without significant loss of titer are near or belowthe levels detectable by Grossman et al., Gene 209: 95-103 (1998).

It was concluded that, contrary to the belief in the previous art, thepresence of small amounts of lactose in some commercial lots of complexmedia is an important factor, and perhaps the determining factor, in thespontaneous induction of target protein synthesis sometimes observed inthe T7 expression system near the onset of stationary phase. Theconclusion seems quite reasonable given that the complex media containtryptic digests of casein (a milk protein), milk contains substantialamounts of lactose, and only very small amounts of lactose are needed tocause induction. Although the casein used in the tryptic digests waspurified in its preparation from milk, it seems almost certain thatsmall amounts of lactose contaminating certain lots of purified caseinare responsible for the inducing activity observed.

The realization that the inducing activity was due to lactose, togetherwith the results of experiments exploring the effects of differentmixtures of glucose and lactose on induction behavior, caused rethinkingof the results and conclusions of Hoffman et al., Protein Expression andPurification 6: 646-654 (1995) on the induction of target protein bybatch-fed lactose or glucose-lactose mixtures under controlledconditions in a fermentor.

It seemed from initial results that cultures grown in different mixturesof glucose and lactose were not inducing production of target protein atintermediate rates, as proposed by Hoffman et al., Protein Expressionand Purification 6: 646-654 (1995), but were in fact growing with littleor no induction until the glucose was depleted, and only then beinginduced by the lactose present in the medium. A comprehensive series oftests, including time courses of growth and induction under differentconditions, were consistent with this interpretation. The termauto-induction is used to refer to the growth pattern of inducibleexpression strains in inducer-containing media, where growth isessentially normal in the early stages, with little or no induction, andexpression of the target protein is turned on automatically at a laterstage of growth, with no intervention. Much of the testing to define theauto-induction behavior and the factors affecting it was done withtarget protein P21 of a structural genomics project, a well induced, notparticularly toxic protein (yeast peptide chain release factorsubunit 1) expressed under control of a T7lac promoter in a pET vectorin B834(DE3) which also contained a compatible plasmid expressing tRNAsfor rare arginine, isoleucine and leucine codons. The results may besummarized as follows.

Little growth was apparent in simple salts media containing lactose assole carbon source, presumably because the level of induction was toohigh to allow growth. Addition of increasing concentrations of glucoseto media containing 0.1% to 1.5% lactose allowed growth to increasingsaturation densities, with expression of target protein occurring incultures that had low glucose concentrations, but decreasing toundetectable levels at about 0.5% glucose or higher. In somewhat of asurprise, ZY-containing media with no added glucose suppressed inductionby even high concentrations of lactose (1.5%) to undetectable levelsduring early log phase growth, followed by high-level induction oftarget protein, typically occurring at an A600 between about 1 and 2.This suppression of induction by lactose in early log phase in theabsence of added glucose is apparently due, at least partly, to thepresence of amino acids rather than, for example, contaminating glucosein the complex ZY media, because purified amino acids also have thissuppressive effect in simple salts media containing lactose andglycerol. Serine seems to be particularly effective in suppressinglactose induction in early log phase, and can itself allow growth andinduction in a lactose-glycerol mixture about as well as a mixture ofall 20 amino acids. Serine is not required, however, as a mixture of 17amino acids lacking serine, cysteine and tyrosine was also effective.

Lactose itself appears not to be a very good carbon source for growth ofauto-induced cultures to high cell densities. Addition of glycerol tosimple or complex media allows growth to much higher densities and doesnot seem to interfere with auto-induction. Mixtures containing from 0.5%to 2.5% glycerol with 0.05% glucose and 0.2% lactose have been veryeffective at promoting growth to high culture densities andauto-induction of many different target proteins in both simple andcomplex media. A short-hand notation for these particular mixtures hasbeen 5052 to designate the mixture 0.5% glycerol, 0.05% glucose, 0.2%lactose, 10052 to designate 1% glycerol, 0.05% glucose, 0.2% lactose,and so on, where the last three digits, 052, denote the 0.05% glucose,0.2% lactose and the first one or two digits refer to the concentrationof glycerol. Other economical and readily available carbon sources thathave been effective in promoting high-density growth and auto-inductionin combination with glucose and lactose include maltose and sorbitol.

Saturation densities of auto-induced cultures can vary considerablydepending on the effect of the expressed target protein on the hostcell. Auto-induced cultures which express target proteins that directlyaffect growth may saturate with high levels of target protein at an A600of around 5. More typical are saturation densities in an A600 range of10-20, and densities as high as 30-50 have been observed. Auto-inductionwith production of large amounts of target protein has been effectivewith many different target proteins at temperatures between 18° C. and37° C.

One factor that significantly affects the saturation density is level ofaeration. This was varied in experiments by changing the volume ofculture relative to the volume of the vessel. Lower relative volumes ofculture provide higher levels of aeration. Both saturation and inductionoccurred at lower cell density, the lower the level of aeration, but thelevel of target protein produced per cell seemed to remain fairlyconstant over a range of saturation densities. Extremely high aerationoccasionally seemed to delay or reduce induction to the point where ithardly occurred, perhaps because an extremely high cell density depletedan essential nutrient, or because some general stress response involvedin high-level induction was muted. Growth of 0.5-ml cultures in 13×100mm glass culture tubes or 500-ml cultures in 2- or 2.8-liter baffledFernbach flasks seem to provide an appropriate level of aeration foreffective auto-induction with media formulations comparable to thosegiven in Table 1.

The auto-induction phenomenon is consistent with a large body ofprevious work showing that the presence of glucose in the growth mediumexcludes the use of lactose both by preventing the uptake of lactosefrom the medium by the lac permease, and through catabolite repression,which operates through the effect of glucose metabolism on cAMP levels.However, the lacUV5 promoter, which directs the expression of T7 RNApolymerase, does not require cAMP-mediated activation and can be inducedby IPTG in the presence of glucose. IPTG acts directly as an inducer anddoes not require the lac permease to enter the cell. Induction bylactose, on the other hand, requires both uptake by the lac permease,and conversion to the true inducer by the transgalactosidation activityof β-galactosidase. Thus, one would not expect auto-induction to work inT7 expression strains that carry mutations in the lac permease becausesuch cells cannot take up lactose, nor in cells that carry mutations inβ-galactosidase that prevent the transgalactosidation reaction whichgenerates the true repressor.

Grossman et al., Gene 209: 95-103 (1998) found that unintended inductionwas reduced in a cAMP-deficient mutant of BL21(DE3), and proposed thatcAMP, rather than acting directly on the lac promoter, may trigger ageneral stress response occurring during the approach to saturation. Itseems quite possible that some such response may be involved in theauto-induction phenomenon.

Catabolite repression and inducer exclusion by the presence of glucosein the growth medium is a general phenomenon in E. coli and is known toaffect other carbon sources besides lactose, including galactose,maltose, arabinose, and many others. Expression systems that usepromoters regulated by any of the compounds subject to cataboliterepression and inducer exclusion would also be suitable for applicationof the auto-induction methods disclosed here.

3. Applications

The current understanding of auto-induction of target protein productionin the T7 system, and the development of reliable media and protocolsfor batchwise growth of expression strains to saturation either withessentially no induction or with auto-induction of high levels of targetprotein at high culture densities, has great utility for producingproteins from cloned coding sequences. Both non-inducing andauto-inducing cultures are simply inoculated and grown to saturation.Non-inducing media produce cultures that are viable for weeks atrefrigerator temperature for making subcultures for screening expressionand solubility or production of substantial amounts of target protein.Agar plates made with non-inducing media also allow expression strainsto be obtained for some target proteins that are too toxic to beobtained in typical commercial media.

In contrast to conventional inductions by addition of either IPTG orlactose, where growth of each culture must be monitored and inducer(IPTG or lactose) added at the proper time, auto-inducing cultures aresimply inoculated and grown to saturation. At 37° C., high-levelauto-induction is usually achieved in 14 hours in minimal salts media,or 8-10 hours in media supplemented with ZY or amino-acid mixtures,convenient lengths of time for overnight inductions. Continuedincubation for several hours after saturation appears not to bedeleterious. In fact, where high levels (typically 2% or more) ofglycerol or other neutral carbon source such as maltose are present andthe pH remains near neutral, cell density can continue increasing slowlyfor 24 hours or longer and can lead to substantial further increases inculture density and yield of target protein. (A potential problem withusing high concentrations of maltose is that most lots have significantcontamination with glucose, which, if high enough, could interfere withinduction.) Auto-induction at lower temperatures, such as 20° C.requires substantially longer incubations, often 24-36 hours. Theincubation time can be shortened without reducing the desired solubilityof the induced target protein by incubating the cultures at 37° C. for afew hours and then transferring them to the lower temperature beforethey become more than lightly turbid.

The densities of induced cells obtained by auto-induction in thedisclosed media are much higher than those typically produced byconventional induction. The densities of batchwise auto-induced culturesare typically high enough that screening for expression and solubilityis accomplished with 5-50 μl of culture, readily obtainable in 96-wellplates. The simplicity and reliability of obtaining non-induced andauto-induced cultures makes highly parallel screening of many targetproteins readily automatable.

Larger-scale growth of auto-induced cultures to obtain protein forpurification is readily accomplished in baffled flasks on a rotoryincubator at 300-350 rpm. A single 1.8- or 2.8-liter baffled Fernbachflask with 500 ml of auto-induced culture can yield tens to hundreds ofmilligrams of target proteins from well expressed clones, sufficient forstructure determination and many other purposes. It is not unusual forauto-induced cultures to yield ten times the amount of purified targetprotein as obtained from the same volume of culture induced with IPTG inthe conventional way.

The disclosed fully defined auto-inducing media also make it possible todevelop media for efficiently labeling proteins. Useful examples areSe-Met labeling for protein structure determination by X-raycrystallography, or isotopic labeling for structure determination byNMR. An efficient auto-inducing medium for Se-Met labeling is thePASM-5052 medium given in Table 1.

To label target proteins with Se-Met, PASM-5052 medium is inoculatedwith a fresh overnight culture grown in PA-0.5G. Growth at 37° C. from athousand-fold dilution into PASM-5052 typically reaches saturation in14-16 hours. Growth at 20° C. is much slower and a culture can take 3days or longer to become induced and reach saturation.

Auto-induction in PASM-5052 medium will produce target proteinsessentially fully labeled with Se-Met when expressed in either themethionine-auxotroph B834(DE3) or the prototroph BL21(DE3). The presenceof Se-Met reduces the growth rate of the two strains comparably,presumably because both strains incorporate Se-Met into their proteinsin place of methionine (but possibly due to other toxic effects aswell). Enzymes of the methionine synthesizing pathway are apparentlyrepressed by the presence of Se-Met in the medium, as they would be bymethionine, preventing endogenous production of methionine.

The small amount of methionine present in PASM-5052 medium allowssignificantly faster growth in the presence of Se-Met, and theconcentration of Se-Met is sufficient to support the growth of themethionine-requiring B834 to saturation (in the absence of vitamin B12).The presence of vitamin B12 in PASM-5052 medium significantly increasesthe yield of target protein and largely prevents the appearance of abrown-orange color that can appear in cells upon continued incubation atsaturation in the presence of a slight excess of Se-Met. Vitamin B12 isknown to activate an enzyme (the product of metH) that methylateshomocysteine to produce methionine. Perhaps a significant fraction ofSe-Met is converted to Se-homocysteine during growth or induction inthis medium, and the B12-dependent methylase stimulates production oftarget protein by regenerating Se-Met.

TABLE 1 Compositions of representative non-inducing and auto-inducingmedia Na₂HPO₄ KH₂PO₄ NH₄Cl (NH₄)₂SO₄ Na₂SO₄ MgSO₄ FeCl₃ 18aa Gluc LactSucc mM mM mM mM mM mM μM metl ZY μg/ml Glyc % % % mM Non-inducingmedia: P-0.5G 50 50 25 1 0.1x 0.5 PA-0.5G 50 50 25 1 0.1x 100 0.5ZYP-0.8G 50 50 25 1 1x 0.8 NIMS 12.5 12.5 50 5 1 0.1x 0.5 20Auto-inducing media: ZYP-5052 50 50 25 1 1x 1x 0.5 0.05 0.2 PA-5052 5050 25 1 1x 200 0.5 0.05 0.2 P-5052 50 50 25 1 1x 0.5 0.05 0.2 PASM-505250 50 25 1 1x ** 0.5 0.05 0.2 MS-15052 12.5 12.5 50 5 2 50 1x 1.5 0.050.2 35 MAS-15052 12.5 12.5 50 5 2 50 1x 200 1.5 0.05 0.2 15 ZYM-1505212.5 12.5 50 5 2 50 1x 1x 1.5 0.05 0.2 ** PASM-5052 contains 200 μg/mleach of 17 amino acids (no M, C, Y), 10 μg/ml methionine, and 125 μg/mlselenomethionine plus 100 nM of vitamin B12 ZY is 1% N-Z-Amine AS + 0.5%yeast extract metl is the metals mix shown in Table 2 Metals may beomitted from media containing ZY if high concentrations are not requiredMetals may be reduced to 0.1x in simple salts media if highconcentrations are not required 18aa gives the concentration of each of18 of the natural amino acids (no cysteine or tyrosine) Glyc is glycerolGluc is glucose Lact is alpha lactose Succ is Na₂ succinate

TABLE 2 Composition of a Trace Metals Mix (1x) 50 μM FeCl₃ 20 μM CaCl₂10 μM MnCl₂ 10 μM ZnSO₄ 2 μM CoCl₂ 2 μM CuCl₂ 2 μM NiCl₂ 2 μM Na₂MoO₄ 2μM Na₂SeO₃ 2 μM H₃BO₃Dilute from a 1000× stock solution in ˜50 mM HCl

1. A bacterial growth medium for suppressing auto-induction ofexpression, in bacterial cells grown batchwise, from a promoter which isrepressed by a lac repressor, selected from the group consisting of0.5G, PA-0.5G, ZYP-0.8G, NIMS and combinations thereof.
 2. The bacterialgrowth medium of claim 1 wherein the promoter is selected from the groupconsisting of a lac promoter, a lacUV5 promoter and a T7lac promoter. 3.The bacterial growth medium of claim 1 wherein the bacterial cellscontain a DE3 lysogen or derivative thereof.
 4. The bacterial growthmedium of claim 3 wherein the bacterial cells are selected from thegroup consisting of Escherichia coli, Bacillus subtilis, Ralstoniaeutrophus, Salmonella enterica serovar Typhimurium, Pseudomonas andRhodobacter capsulatus.
 5. The bacterial growth medium of claim 4wherein the cells are Escherichia coli.
 6. The bacterial growth mediumof claim 5 wherein the E. coli cells are selected from the groupconsisting of BL21(DE3), B834(DE3), HMS174(DE3), and derivativesthereof.
 7. The bacterial growth medium of claim 3 wherein the bacterialcells further contain a target DNA in a T7 plasmid expression vector,which target DNA optionally comprises a coding sequence for a targetprotein.
 8. The bacterial growth medium of claim 7 wherein the targetDNA is under the control of a T7 RNA polymerase promoter.
 9. A bacterialgrowth medium for suppressing auto-induction of expression, in bacterialcells grown batchwise, from a promoter which is repressed by a lacrepressor comprising 0.5G, PA-0.5G, ZYP-0.8G, NIMS or combinationsthereof.